Correction: Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses.

[This corrects the article DOI: 10.1371/journal.pntd.0002157.].

Dobrava virus carried by the yellow-necked field mouse Apodemus flavicollis, causing hemorrhagic fever with renal syndrome in Romania.

Hemorrhagic fever with renal syndrome (HFRS) has been confirmed by serological methods during recent years in Romania. In the present study, focus-reduction neutralization tests (FRNT) confirmed Dobrava hantavirus (DOBV) as the causative agent in some HFRS cases, but could not distinguish between DOBV and Saaremaa virus (SAAV) infections in other cases. DOBV was detected by a DOBV-specific TaqMan assay in sera of nine patients out of 22 tested. Partial sequences of the M genomic segment of DOBV were obtained from sera of three patients and revealed the circulation of two DOBV lineages in Romania. Investigation of rodents trapped in Romania found three DOBV-positive Apodemus flavicollis out of 83 rodents tested. Two different DOBV lineages were also detected in A. flavicollis as determined from partial sequences of the M and S genomic segments. Sequences of DOBV in A. flavicollis were either identical or closely related to the sequences obtained from the HFRS patients. The DOBV strains circulating in Romania clustered in two monophyletic groups, together with strains from Slovenia and the north of Greece. This is the first evidence for the circulation of DOBV in wild rodents and for a DOBV etiology of HFRS in Romania.

Indirect immunofluorescence assay for the simultaneous detection of antibodies against clinically important old and new world hantaviruses.

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n=97); SEOV (China, n=5); DOBV (Romania, n=7); SNV (Canada, n=23); ANDV (Argentina and Chile, n=52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n=177; 96%) and/or IgM (n=131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).

First serologic evidence for the circulation of Crimean-Congo hemorrhagic fever virus in Romania.

Serum samples from sheep in localities situated in the county of Tulcea, Northern Dobrogea, were tested with an IgG sandwich ELISA using a recombinant Crimean-Congo hemorrhagic fever virus (CCHFV) antigen. In all, 131 sera out of 471 tested (27.8%) had IgG antibodies specific to CCHFV. This is the first evidence for the circulation of CCHFV virus in Romania.

Hantavirus infection--hemorrhagic fever with renal syndrome: the first case series reported in Romania and review of the literature.

Hemorrhagic fever with renal syndrome (HFRS) is a zoonotic infectious disease caused by Hantaviruses, a group of RNA viruses belonging to the Bunyaviridae family. Humans may get the disease by contamination with excreta of carrier rodents. The disease typically manifests with the triad fever-thrombocytopenia-acute kidney injury (AKI). Although its global prevalence seems to be increasing, Hantavirus infection is still commonly overlooked, because of its clinical polymorphism and non-specific symptoms, particularly in mild cases. Until recently, the disease was virtually unknown in Romania, due to lack of physicians' awareness and of adequate laboratory diagnostic techniques. In this article, we present the first six cases of HFRS diagnosed in our country, based on serology testing. We review the existing literature on HFRS and discuss our findings in comparison with other reports. All our patients presented with fever, flu syndrome, bleeding, gastrointestinal symptoms, and oliguria. Among laboratory abnormalities, elevated serum creatinine and liver enzymes, high C-reactive protein, leukocytosis, low platelet count, and hematuria were constantly seen. Five patients required hemodialysis. All patients survived and five of them completely recovered their renal function, while only one patient retained a mild impairment of the glomerular filtration rate. From a clinical viewpoint, we believe that Hantavirus infection should be considered in all patients presenting with fever, thrombocytopenia, and AKI, when specific serology testing would be indicated. From a public health perspective, we suggest that future efforts in our country should be directed toward (1) increasing the understanding and the awareness of this disease among health care professionals, (2) educating the population at risk on the application of prophylactic measures, (3) expanding the availability of diagnostic laboratory tools, and (4) developing research on national zoonotic virus reservoirs.